The objective of this study is to reveal the molecular nature of the apparently distinct germ cell adenylate cyclase system and to clarify its role in sperm function. During spermiogenesis Mn 2 ion-sensitive enzyme activity was found in the cytosol, as well as in the particulate subcellular fraction derived from rat testis. In testis cell fractions enriched in specific germ cell types the relative levels of the soluble and particulate enzyme activities were found to be different. In cell fractions enriched in early and late spermatids the proportion of the cytosol enzyme was greater, while in cell fractions enriched in testis sperm the proportion of the particulate enzyme was greater than the level of the soluble enzyme. In spermatozoa obtained from the epididymis the enzyme was found exclusively particulate. The question whether the testis cytosol and the membrane-bound enzymes in the testis and epididymal sperm are the same or separate enzyme entities will be tested. The kinetic properties of the cytosol and particulate enzyme will be compared, and their immunochemical homology or distinctivness will be determined. The testis cytosol enzyme has been recently highly purified. In continuation of these studies the purified enzyme will be used to prepare specific antibodies. Antibodies to the highly purified enzyme will be used for immunohistochemical localization in situ of the enzymes during spermatogenesis. The adenylate cyclase specific antibodies will be used to assertain the immunogenic specificity of the germ cell enzyme. In attempting to define the role of this particular enzyme in sperm function, studies are proposed to identify and characterize the protein targets for endogenous phosphorylation, and thus define the molecular mode of action of cyclic AMP in spermatozoa through its effect on regulatory and/or contractile proteins.